Objectives: To observe the effect of electroacupuncture(EA) on endometrial fibrosis and M1-type macrophages in rats with intrauterine adhesions(IUA), so as to explore the possible mechanism of EA in the treatment of IUA.
Methods: Fifteen female SD rats were randomly divided into blank group, model group and EA group, with 5 rats in each group. The IUA rat model was established by double damage method using mechanical scraping combined with lipopolysaccharide infection. Rats in the EA group were treated with acupuncture at "Guanyuan"(CV4), and EA at bilateral "Zusanli"(ST36) and "Sanyinjiao"(SP6)for 20 minutes each time, once a day, for 3 consecutive cycles of estrus. Five rats in each group were sampled during the estrous period, and the endometrial morphology, endometrial thickness and the number of blood vessels and glands were observed after HE staining. The fibrotic area of the uterus was observed after Masson staining. The positive expressions of Runt-related transcription factor(RUNX1), transforming growth factor-β1(TGF-β1), connective tissue growth factor(CTGF), α-smooth muscle actin(α-SMA), collagen type I(Col-Ⅰ), cluster of differentiation 86(CD86), interleukin-1β(IL-1β), and tumor necrosis factor-α(TNF-α) in endometrial tissue were detected by immunohistochemistry. Western blot was used to detect relative protein expressions of RUNX1, TGF-β1, α-SMA, CD86, and TNF receptor 2 (TNFR2), and real-time fluorescence quantitative PCR was used to detect mRNA expressions of RUNX1, TGF-β1, α-SMA, CD86, and TNF-α in the endometrium.
Results: During the estrous phase, the endometrial layer in the model group was damaged, with reduced folds, disordered arrangement of epithelial cells, loose fibrous connective tissue, significant narrowing and adhesions in the uterine cavity, interstitial congestion, edema, and a significant infiltration of inflammatory cells with sparse glands. While uterine tissue structure of the EA group was basically intact, resembling a normal uterus, with more newly formed glands and a small amount of inflammatory cell infiltration. In comparison with the blank group, the endometrial thickness, the number of blood vessels, and the number of glands were significantly decreased(P<0.001) in the model group, while the ratio of uterine fibrosis area, the positive expressions of RUNX1, TGF-β1, CTGF, α-SMA, Col-Ⅰ, CD86, IL-1β, and TNF-α, the protein relative expressions of RUNX1, TGF-β1, α-SMA, CD86 and TNFR2, and the mRNA relative expression levels of RUNX1, TGF-β1, α-SMA, CD86 and TNF-α in the endometrium were significantly increased (P<0.001, P<0.01). Compared to the model group, the endometrial thickness, the number of blood vessels, and the number of glands were significantly increased(P<0.01, P<0.05) in the EA group, while the ratio of uterine fibrosis area, the positive expressions of RUNX1, TGF-β1, CTGF, α-SMA, Col-Ⅰ, CD86, IL-1β and TNF-α in the endometrial tissue, the protein expressions of RUNX1, TGF-β1, α-SMA, CD86 and TNFR2, and the mRNA relative expressions of RUNX1, TGF-β1, α-SMA, CD86 and TNF-α in the endometrium were significantly decreased (P<0.001, P<0.01, P<0.05).
Conclusions: EA can improve endometrial fibrosis in IUA rats, which may be related to its function in decreasing the level of endometrial M1-type macrophages and the secretion of related inflammatory factors.
目的: 观察电针对宫腔粘连（IUA）大鼠子宫内膜纤维化的影响，并以M1型巨噬细胞为切入点，探讨电针治疗IUA的可能机制。方法: 雌性SD大鼠随机分为空白组、模型组、电针组，每组5只。采用机械搔刮联合脂多糖感染双重损伤法建立IUA大鼠模型。电针组予“关元”针刺、“足三里”“三阴交”电针干预，20 min/次，1次/d，连续干预3个动情周期。每组大鼠于动情期取材，HE染色法观察大鼠子宫内膜形态、子宫内膜厚度及血管、腺体数目，Masson染色法观察子宫纤维化面积，免疫组织化学法检测子宫内膜组织中Runt相关转录因子（RUNX1）、转化生长因子-β1（TGF-β1）、结缔组织生长因子（CTGF）、α平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原蛋白（Col-Ⅰ）、分化簇86（CD86）、白细胞介素-1β（IL-1β）、肿瘤坏死因子-α（TNF-α）的阳性表达，Western blot法检测子宫内膜组织中RUNX1、TGF-β1、α-SMA、CD86、肿瘤坏死因子受体2（TNFR2）的蛋白相对表达量，实时荧光定量PCR法检测子宫内膜组织中RUNX1、TGF-β1、α-SMA、CD86、TNF-α的mRNA相对表达量。结果: 模型组大鼠动情期子宫内膜层被破坏，皱襞减少，上皮细胞排列紊乱，纤维结缔组织疏松，宫腔明显狭窄并出现粘连，间质充血、水肿，可见较多的炎性细胞浸润，腺体稀疏；电针组大鼠子宫组织结构基本完整，接近于正常子宫，可见较多新生腺体，少量炎性细胞浸润。与空白组相比，模型组子宫内膜厚度、血管数目及腺体数目显著减少（P<0.001）；子宫内膜纤维化面积比值，子宫内膜中RUNX1、TGF-β1、CTGF、α-SMA、Col-Ⅰ、CD86、IL-1β、TNF-α阳性表达的平均吸光度，子宫内膜组织中 RUNX1、TGF-β1、α-SMA、CD86、TNFR2蛋白表达，RUNX1、TGF-β1、α-SMA、CD86、TNF-α mRNA相对表达量显著升高（P<0.001，P<0.01）。与模型组相比，电针组子宫内膜厚度、血管数目、腺体数目显著增加（P<0.01，P<0.05）；子宫内膜纤维化面积比值，子宫内膜中RUNX1、TGF-β1、CTGF、α-SMA、Col-Ⅰ、CD86、IL-1β、TNF-α阳性表达的平均吸光度值，子宫内膜组织中RUNX1、TGF-β1、α-SMA、CD86、TNFR2蛋白表达，RUNX1、TGF-β1、α-SMA、CD86、TNF-α mRNA相对表达量显著下降（P<0.001，P<0.01，P<0.05）。结论: 电针能够降低IUA大鼠子宫内膜M1型巨噬细胞的水平，减少M1型巨噬细胞相关炎性因子分泌，从而改善IUA大鼠子宫内膜纤维化。.
Keywords: Electroacupuncture; Endometrial fibrosis; Inflammations; Intrauterine adhesions; M1-type macrophages.